tPA is the abbreviation adopted in the art for a tissue plasminogen activator, i.e., the 2-chain type of plasminogen activator which has been identified for example in Bowes melanoma cells. Pro-tPA is the abbreviated name given to the precursor of tPA, i.e., a single chain compound having the same molecular weight as tPA and which by certain enzymes is converted into double chain tPA. According to the prior art literature, the single- and double-chain forms were said to both have virtually identical activity. The present invention will be shown to involve a radical departure from that view.
Plasminogen activators are substances which by their action upon plasminogen (a precursor of plasmin) result in the formation of plasmin. Plasmin in turn acts upon fibrin (or blood clots) to liquefy or dissolve the fibrin. Plasmin also causes lysis of fibrinogen which is a precursor of fibrin.
The aforegoing effects play an important role in the natural fibrinolytic systems. They are also put to use in the therapeutic administration of plasminogen activators for the management of thrombotic disease or other conditions where it is desirable to produce local fibrinolytic or proteolytic activity via the mechanism of plasminogen activation.
They may also find use in reagents for diagnostic, pathological or scientific tests involving fibrinolysis in vitro.
Two activators of human plasminogen are extensively used. The first of these is the bacterial protein, streptokinase, which functions by a non-proteolytic mechanism. The second is the protease, urokinase, which is obtained from urine or cultured kidney cells, although it is also known to occur in most other kinds of mammalian tissue. These two compounds have the therapeutic disadvantage that their action is not confined to plasminogen associated with fibrin in blood clots. They act upon plasminogen generally in the circulation and, in consequence, produce widespread plasmin generation with extensive lysis of fibrinogen, the precursor of fibrin. This may in turn lead to a bleeding state as a result of ineffective normal coagulation of the blood. Furthermore, urokinase has had the disadvantage that it is costly to isolate and prepare and is obtained in poor yields by the processes currently in use. This disadvantage also applies to valuable uses of urokinase other than fibrinolytic therapy, and restricts the availability of urokinase as a valuable commercial product for such uses. Streptokinase,being a foreign protein, elicits an immune response from the patient. The resulting antibodies neutralise the action of streptokinase on plasminogen and hence diminish its therapeutic efficacy.
A third plasminogen activator referred to generally as "tissue plasminogen activator" hereinafter referred to as "tPA" is also known to exist. This enzyme is found in most human tissues and is identical to or indistinguishable from an enzyme that is also a characteristic secretory product of human melanoma cells cultured in vitro. Although it catalyzes the proteolytic activation of plasminogen in much the same way as does urokinase, tPA differs from urokinase in a number of important respects. The two enzymes are chemically dissimilar. They have different molecular weights and each fails to react with antibodies to the other enzyme. The catalytic action of tPA is enhanced by fibrin whereas that of urokinase is not. tPA has the important property that it binds to fibrin, whereas urokinase fails to do so. These facts are recorded in a recent article entitled "Purification and characterisation of the plasminogen activator secreted by human melanoma cells in culture" by C Dingeman C. Rijken et alia (J. of Biological Chem., 256, No 13, pages 7035 to 7041). That same article also describes the preparation of tPA from a cultured human melanoma cell line as well as from normal human tissue. The purification procedure consisted of successive chromatography on zinc chelate-agarose, concanavalin A-agarose and sephadex G-150 in the presence of detergent.
The tendency for tPA to bind to fibrin, its greatly enhanced fibrinolytic action in the presence of fibrin and when compared to urokinase and streptokinase, its relatively inefficient function as a plasminogen activator in the absence of fibrin combine to make tPA a plasminogen activator of choice for human thrombolytic therapy. Interactions between fibrin and tPA to a considerable extent localise the plasmin generation to the site of the clot and mitigate or avoid the consequences of promiscuous plasminogen activation as observed when urokinase or streptokinase is used. Furthermore streptokinase is a protein which is immunochemically foreign to man, whereas tPA is not.
A recent report entitled "Specific lysis of an iliofemoral thrombis by administration of extrinsic (tissue type) plasminogen activator" by W. Weimar et alia (Lancet, Nov. 7, 1981, page 1018) testifies to the clinical usefulness of tPA in the management of human thrombotic disease.
According to P. Wallen et al. (Prog. Chem. Fibrinolysis Thrombolysis 5, 16-23 (1981)) tPA occurs in two different forms, one being a single-chain form which is converted by plasmin or trypsin into two chains linked by disulphide bridges, and which those authors consider to be a degradation product of the former. The two-chain form is said to be fibrinolytically more efficient in fibrinolytic test systems (in vitro) than the one-chain form. Aprotinin is said to inhibit the conversion of the one-chain into the two-chain form so that theone-chain form may be recovered as the only or predominating form.
This work is referred to in more recent publication by Dingeman, Rijken, Hoylaerts and Collen (J. Biol. Chem., 257, 2920-2925 (1982), European patent application 0 041 766) who describe both forms as plasminogen activators, whose "plasminogen activating properties . . . are similar". They suggest that the one chain form when it is adsorbed on a fibrin clot is quickly converted to the two-chain form so that the fibrinolysis "occurs mainly via two-chain derivative". They conclude: "this conversion does not seem to play a role in the regulation of fibrinolysis". According to their findings the "two molecular forms had the same activity (lysistime.)", and "the catalytic efficiencies of both molecular forms are virtually identical therefore the one-chain form cannot be considered to be a less active precursor nor the two-chain form to be a less active degradation product". Whether or not the aforesaid conclusions are all correct, it is clear that both "molecular forms" have utility for therapeutic as well as in vitro fibrinolysis.
In actual fact, in the light of recent research results by the present inventors, it is clear that the above conclusions require some qualification. Pro-tPA is indeed in a sense a precursor of tPA, although not a totally inactive precursor. Its inherent activity is about one tenth that of tPA, so that on that basis alone it would at first sight appear preferable to administer tPA for therapeutic purposes rather than pro-tPA which first has to be converted into tPA before it can exercise its full therapeutic effect.
As a matter of fact, although European patent application 0041766 describes two compounds which presumably represent tPA as well as pro-tPA, only the former compound is actually characterized in respect of its fibrinolytic, fibrinogenolytic and thrombolytic effects. There is no such description for pro-tPA, nor any specific description of its use as a pharmaceutical composition. The claims for the pharmaceutical use of the compounds, to the extent that they are specific are directed only to the double-banded form, i.e., tPA. The only published actual therapeutic use of the substance has been the use in the double-stranded tPA form (Weimer et al., The Lancet, Nov. 7, 1981 p 1018-1020).
According to the aforesaid prior art, no method was known which lends itself readily to the manufacture of 2-chain tPA on a commercial scale, nor of the single-chain form herein referred to as "pro-tPA".
According to a recent proposal by the present inventors (RSA patent application 82/9168 and applications based thereon in other countries and which at present are not yet part of the state of the art) a process has been proposed which lends itself readily to such commercial scale manufacture.